The beneficial effects of conjugated long chain polyunsaturated fatty acids in food products for animals or humans have been recognised in the prior art.
EP 411.101 e.g. discloses, that compositions containing free conjugated linoleic acid (=CLA), such as 9.11-dienic and 10.12-dienic fatty acids or non-toxic salts thereof can be used to preserve products by inhibiting mould growth. According to this EPxe2x80x2 101 the free acids are prepared by reacting linoleic acid with a protein, capable of effecting the transformation of linoleic acid to the desired acid forms at temperatures up to 85xc2x0 C. The CLA obtained contains both the 9.11 and 10.12-octadecadienoic acids and active isomers therefrom. Because of cis/trans-isomerism above CLA""s can contain 8 different isomers, i.e. cis9 -cis11; cis9-trans11; trans9-cis11; trans9-trans11; cis10-cis12; cis10-trans12; trans10-cis12 and trans10-trans12. From those isomers the cis9-trans11 and trans10-cis12 are the most abundant, while their concentrations are about equal. It is generally believed, that those two most abundant isomers are responsible for the beneficial effects of the compositions, containing CLA""s.
According to EP 440.325 CLA""s can be applied as xe2x80x9cmetal chelatorxe2x80x9d in natural foods. The CLA""s contain 9.11 and 10.12-octadecadienoic acid, salts or other derivatives thereof. The free acids can be prepared by e.g. an enzymic treatment, using xcex9412 cis xcex9411 trans isomerase, of linoleic acid.
In U.S. Pat. No. 5,430,066 it is disclosed, that CLA""s can be applied in foods for preventing weight loss, reduction in weight gain or anorexia in animals or humans. Also disclosed is, that these CLA""s can alleviate the adverse catabolic effects of a product from the immune-system, in particular from interleukin-1.
From U.S. Pat. No. 5,428,072 it is known, that CLA""s can be used for the increase of the efficiency of feed conversion to body weight in an animal.
Shantha c.s disclosed in J. of AOAC Intern 76 (3) 1993, p. 644-649 that CLA-isomers are potential anticarcinogens.
According to Fogerty c.s in Nutrition Reports Intern 38 (5) , 1988, p. 937-944 cis9-trans11 linoleic acid can be used in various foods or human milk.
U.S. Pat. No. 4,164,505 discloses a process, wherein unconjugated unsaturated fatty acids are isomerised into conjugated unsaturated fatty acids by a treatment with base. As a result of this process a kinetically controlled reaction-mixture will be obtained, wherein the double bonds are conjugated but distributed over the whole carbon chain of the polyunsaturated fatty acids. Therefore this process does not result in organic materials, wherein the two most abundant conjugated polyunsaturated fatty acid moieties L1 and L2 are present in a weight-ratio                     L        1                    L        2              =          2.3      -      99        ,
as we aim for as a result of our process.
Above prior art methods and products do have a number of drawbacks. E.g. the methods for the preparation of the CLA""s according to above prior art cannot be applied on a commercial scale, e.g. because the yields of the products are very limited. Moreover the products obtained always will have one specific ratio between the cis9-trans11/trans10-cis12 isomers (in general about 1.0). Therefor compositions with an other ratio than 1.0 cannot be obtained. As the effectiveness of the two isomers for specific purposes are different it is highly desirable to have the opportunity to make CLA""s, wherein the ratio             cis      9        -          trans      11                  trans      10        -          cis      12      
can be chosen freely, depending on the conditions applied during the process.
Therefore our invention concerns a new process for the preparation of CLA""s, wherein the ratio             cis      9        -          trans      11                  trans      10        -          cis      12      
can be chosen freely. This new method can be applied for the preparation both of new CLA-compositions and known CLA-compositions.
So our inventions concerns a process for the preparation of materials, containing conjugated unsaturated fatty acid moieties, wherein a material, containing at least 5 wt % of conjugated polyunsaturated fatty acid moieties, comprising at least two different isomers L1 and L2 in a weight ratio L1:L2=XA, is subjected to an enzymic conversion, selected from one of the following conversions:
(i) free fatty acids with:
(a) mono- or polyalcohols, or
(b) mono, -di-triglycerides, or
(c) alkylesters, or
(d) phospholipids
(ii) mono, -di- or triglycerides with:
(a) water, or
(b) mono- or polyalcohols, or
(c) alkylesters, or
(d) phospholipids
(iii) phospholipids with:
(a) water, or
(b) alkylesters, or
(c) other phospholipids, or
(d) mono- or polyols
(iv) alkylesters, or wax-esters with:
(a) water, or
(b) mono- or polyols, or
(c) free fatty acids, or
(d) phospholipids,
wherein an enzyme is applied, that has the ability to discriminate between L1 and L2, which conversion results in a mixture of at least two products (I) and (II), from which at least one product (I) or (II) contains L1 and L2 in a weight-ratio XB, XB being at least 1.1 XA, preferably at least 1.2 XA, most preferably at least 1.3 XA, and wherein L1 and L2 are different isomers of polyunsaturated fatty acids with at least two unsaturations and at least 18 carbon atoms.
Enzymes that can be applied for the enzymic conversion are e.g. Geotrichum candidum and Candida rugosa and phospholipases.
As indicated above many different types of reactants can be applied for the enzymic conversion. It was found, that very good results are obtained, when the conversion is performed on a mixture of free fatty acids, containing at least 5 wt %, preferably at least 10 wt %, most preferably at least 15 wt % of conjugated polyunsaturated fatty acids and a phospholipid or a mono, -di- or triglyceride.
Preferred starting materials, applicable in the process according to the invention have a weight ratio XA (ie L1:L2) of about 1.0.
According to another embodiment of the invention water or glycerol, mixed with a mono, -di- or triglyceride could be converted as well. In this instance the glyceride material is the reactant having at least 5 wt % conjugated polyunsaturated fatty acids in it.
Although above process can be applied on any starting material, wherein L1 and L2 can be chosen from all long chain polyunsaturated fatty acid moieties with at least two unsaturations and 18 or more carbon atoms, as long as the long chain polyunsaturated acids present are present in different cis/trans-isomeric forms, it is preferred that L1 and L2 are cis9 trans11 and trans10 cis12-linoleic acid (or vice versa)
The process of the invention can be applied for the preparation of known compounds, however also novel compositions can be obtained by using this process. These novel compounds (compositions) have unexpected properties, because of the weight-ratio L1:L2 that occurs in these compositions. Therefore our invention also concerns novel organic materials, which materials contain at least 1 wt % of conjugated polyunsaturated fatty acid moieties with a chain length of at least 18 C-atoms, wherein the conjugated polyunsaturated fatty acid moieties at least comprise two isomers L1 and L2 in a weight-ratio:             L1      L2        =          2.3      -      99        ,
preferably 4-20, most preferably 8-15 L1 being the most abundant and L2 being the second most abundant conjugated polyunsaturated fatty acid moiety in the material, while L1 and L2 are different isomers of polyunsaturated fatty acids with at least two unsaturations and at least 18 carbon atoms.
The organic materials, that can be obtained can be: either a mixture of free fatty acids, a mixture of wax-esters, a mixture of low alkylesters, a mixture of monoglycerides, or diglycerides or triglycerides or mono, -di- and triglycerides, or a mixture of phospholipids, or a mixture of one or more components of said mixtures.
In the novel organic materials L1 and L2 can both be selected from cis9,trans11 and trans10,cis12-linoleic acid.
In many instances the starting material for our process will be an animal-derived material, such as a fish oil. However it is also possible to use vegetable oils as starting material. By using such vegetable oils the products of the conversion are novel over any product known in the prior art, as vegetable oils contain small amounts of specific components, which are not present in e.g. the fish oils, and which are indicative for the vegetable source the oil is derived of. So organic materials, derived from vegetable oils, having at least two conjugated polyunsaturated fatty acids moieties L1 and L2, wherein L1 is the most abundant and L2 is the second most abundant conjugated polyunsaturated fatty acid moiety, wherein L1 and L2 are present in a weight-ratio of 1.5-25, preferably 4-20, most preferably 8-15, while the total amount of conjugated polyunsaturated fatty acid moieties in the organic material is at least 1 wt %, and wherein L1 and L2 are different isomers of polyunsaturated fatty acids with at least two unsaturations and at least 18 carbon atoms, are considered to be novel over any prior art product, derived from a non-vegetable source.
As is well-known from the prior art organic materials containing large amounts of polyunsaturated fatty acids are very sensitive for oxygen. Therefore we prefer to add an effective amount of an oxidation stabilizer, selected from the group, consisting of: natural or synthetic tocopherols, TBHQ, BHT, BHA, free radical scavengers, propylgallate, ascorbylesters of fatty acids and enzymes with anti-oxidant properties.
Although our organic materials could be applied as such, it is often preferred to use them as a blend with a complementary fat. Therefore our invention also concerns blends of an organic material and a complementary fat, wherein the blend comprises:
0.3-95 wt % , preferably 2-80 wt %, most preferably 5-40 wt % of the organic material, obtainable by the process according to claims 1-6, or the organic material according to claims 7-11, and
99.7-5 wt %, preferably 98-20 wt %, most preferably 95-60 wt % of a complementary fat, selected from: cocoa butter, cocoa butter equivalents, palm oil or fractions thereof, palmkernel oil or fractions thereof, interesterified mixture of said fats or fractions thereof, or liquid oils, selected from: sunflower oil, high oleic sunflower oil, soybean oil, rapeseed oil, cottonseed oil, fish oil, safflower oil, high oleic safflower oil, maize oil and MCT-oils.
Above blends of organic material and complementary fat preferably display a solid fat content (NMR-pulse, unstabilised) of 0-85, more preferably 10-70, most preferably 20-60 at 5xc2x0 C. and  less than 30, more preferably  less than 20, most preferably  less than  at 35xc2x0 C.
Part of the invention are also food products and animal feed, containing a fatphase, wherein the fatphase contains an effective amount of the product, obtainable by the process of claims 1-5 or the organic material of claims 6-10, or the blend of claims 11-12. The food products are suitably selected from the group consisting of: spreads, margarines, creams, dressings, mayonnaises, ice-creams, bakery products, infant food, chocolate, confectionery, sauces, coatings, cheese and soups.
However also food supplements and pharmaceutical products can be obtained by using our fats or blends. Therefore foodsupplements or pharmaceutical products, that are in the form of capsules or other forms, suitable for enteral or parenteral application and that comprise a product obtainable according to the process of the invention or an organic material or a blend, according to the invention, are also part of the invention.
Analytical Methods
Fatty acid compositions were determined by fatty acid methyl ester gas chromatography (FAME GC) using the method given in JAOCS Vol 71 no 12 page 1321.
Partial glyceride contents were determined by silica gel high performance liquid chromatography (HPLC) using an evaporative light scattering detector with 12, hydroxy iso-octane as an internal standard.
Free fatty acid contents were determined by titration against standard sodium hydroxide and are expressed as % oleic acid.